Imagem electron multiplier ccd camera

10 µl of saturated E. coliOP50 was spotted to an NGM plate and allowed to dry. Fifteen 2-d-old unc-64(lf) hermaphrodites were transferred to the OP50 and allowed to incubate for at least 1 hr. The OP50 + NGM + hermaphrodites were then placed on a microscope slide. The behavior of one male at a time was recorded using an Olympus BX51 microscope and Hamamatsu ImagEM Electron multiplier (EM) CCD camera. Males were given 10 min to insert their spicules into the hermaphrodite uterus, after which the mating trial was terminated. Spicule insertion % was determined by calculating the number of males that were successfully able to insert their spicules within 10 min vs. the total number of males tested. Sperm release from the valve % was determined using the total number of males that inserted. All males that were able to release some sperm from the valve were counted as successful whether or not they were able to release sperm from the cloaca. Sperm release measures all males that successfully transferred sperm into the uterus vs. the total number of males that inserted their spicules. The total amount of time that males’ spicules were inserted measures from when they inserted their spicules until they either retracted their spicules into their cloaca or removed the still-protracted spicules from the uterus. The competition assay was done as described in LeBoeuf et al. (2011) (link).

Worms were isolated by gender at L4 the day before and allowed to mature overnight. One worm was then placed on a fresh OP50E. coli lawn and recorded using an Olympus BX51 microscope and Hamamatsu ImagEM Electron multiplier (EM) CCD camera.

The morning of the experiment, 10 µl of a saturated E. coli OP50 culture was spotted on an NGM agar plate and allowed to dry. 15 two-days-old unc-64(lf) hermaphrodites were transferred to this plate and allowed to incubate for at least 1 hr, to ensure the males would respond to the hermaphrodites. The E. coli + hermaphrodites section of the plate was cut out and placed on a microscope slide and transferred to an Olympus BX51 microscope. Recordings were then made using a Hamamatsu ImagEM Electron multiplier (EM) CCD camera (Japan) of the male mating. Each recording was then analyzed for the mating behavior of interest.